PROJECT SUMMARY/ABSTRACT An estimated more than ninety-five percent of potential Alzheimer?s Disease (AD) therapeutics and prophylactics have extremely limited ability to migrate from circulation to brain tissue. These blood-to-brain transport limitations necessitate unfeasibly high doses of systemically administered drug to realize beneficial effects within the brain and/or promote off-target side effects throughout the body and prevent such transport- impaired molecules from being viable AD drug candidates. Development of a generalizable delivery technology that both targets transport-impaired drugs to the blood brain barrier (BBB), a tightly packed layer of endothelial cells surrounding the nutrient-supplying blood vessels that radiate throughout the brain, and facilitates transport of these drugs across the BBB would dramatically expand our armamentarium of agents for AD therapy and prophylaxis. Such expansion could transform the way clinicians seek to treat and prevent AD by providing the breadth of pharmaceutical options needed to enable development of personalized AD treatment and prevention programs through evaluation of patient responses to different drug combinations. Conjugation of small molecule drug-loaded liposomes or protein drugs to ?Trojan Horse? antibodies (Abs) that both bind to proteins on and are transported across the BBB has been a highly pursued strategy for targeting AD drugs to the central nervous system (CNS). Such Trojan Horse Abs however, bind to proteins expressed on both the BBB and many other tissues throughout the body; this ubiquitous expression results in less than one percent of systemically injected Trojan Horse antibody-drug conjugate doses reaching the brain. In this research project our team will address the above alluded to need for step change improvements in AD drug delivery by simultaneously identifying proteins and/or epitopes that are specific to or highly enriched on BBB endothelial cells (brain microvascular endothelial cells - BMECs) and generating human fibronectin domains (Fn3s), antibody-like biomolecule-binding proteins that can possess exceptionally high target binding affinity and specificity but are less time consuming and expensive than Abs to produce, that bind to these BBB- specific molecular entities and can be superior substitutes for existing Trojan Horse Abs in targeting AD drugs to the CNS. Our innovative adaptation of extrusion techniques employed in household cleaner manufacturing to convert BMECs into water soluble nanometer-sized vesicles, known as CytoBits, brings both novelty and feasibility to this research initiative and uniquely positions us to succeed where others have encountered difficulties in seeking to engineer highly specific BBB-binding drug carrier proteins. Unlike whole cells, CytoBits are compatible with high-throughput cell enrichment methods, which respectively utilize magnetic-microspheres and multicolor fluorescence activated cell sorting (FACS), that will enable us to screen a 250 million clone yeast-displayed Fn3 library and separate BMEC-binding clones from Fn3s that bind to cells derived from nontarget, e.g., lung and cardiac, tissues with fidelity that cannot be approached by the cell panning methods used in drug carrier protein development efforts reported by other groups. Leading candidate BBB-specific drug carrier Fn3s enriched via yeast-displayed Fn3 library screening will be expressed as soluble proteins and their affinities and specificities toward intact BMECs quantified using flow cytometry-based binding assays. Coupling binding assay outcomes with the results of assays measuring rates at which Fn3s are endocytosed by BMECs and utilization of tandem mass spectrometry (MS/MS) to elucidate the identities of BMEC proteins immunoprecipitated using drug carrier candidate Fn3s will yield a dataset that will be holistically considered in choosing up to three BBB-specific Fn3s for evaluation in mouse biodistribution studies carried out after this project?s two-year performance period. Our long-term objective for this CNS drug targeting research initiative is to see BBB-specific drug carrier Fn3s broadly deployed in personalized AD drug programs by the year 2030; our team possesses both the animal study expertise and clinical connections needed to realize this timetable for translation. We are eager to continue our progress toward achieving this goal and are excited about the impact that its realization will have in making a difference for the millions of AD patients and patient families whose lives have been tragically rearranged by this debilitating condition.